Fig. 3: Vitamin B12 supplementation enhances H3K36me3 and has a cell-autonomous role in reprogramming.
From: Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair
a, In vitro reprogramming of MEFs with doxycycline-inducible OSKM for 10 d in the presence of doxycycline (OSKM) and/or vitamin B12, MAT2Ai, KDM4A/KDM4B inhibition (NSC), or SAM as indicated, cultured in KSR. iPS cell colonies were quantified by alkaline phosphatase staining (left) and representative images are shown (right). Each data point represents MEFs generated from an independent embryo (n = 6 OSKM, B12; n = 5 B12 + MAT2Ai, SAM; n = 3 NSC). b, Fraction of total intracellular SAM 13C-labelled at the methyl m + 1 position (Extended Data Fig. 6d), using 13C-serine as a precursor. Labelling was initiated at t = 72 h for 6 h. Data from n = 3 independent MEFs are shown. c, H3K36me3 dynamics during in vitro reprogramming. MEFs were treated with doxycycline with or without vitamin B12 as indicated. A representative immunoblot and quantification from n = 3 independent MEFs are shown. d, H3K36me3 level correlates with reprogramming efficiency in vitro. MEFs were treated as indicated and H3K36me3 levels were probed in histone extracts at day 3 after doxycycline treatment. A representative blot from n = 2 independent MEFs with similar results is shown. e,f, Expression of the H3K36 trimethyl-transferase Setd2 during in vitro (e) and in vivo pancreatic (f) reprogramming. In e, P values represent significant change from the parental MEFs (n = 3 MEFs). In f, samples were collected from WT (n = 4; 3 M 1 F) or OSKM (n = 8; 4 M 4 F) mice after 7 d of doxycycline treatment. g, H3K36me3 during in vivo reprogramming. Pancreatic tissue from OSKM mice treated with doxy (n = 5; 2 M 3 F) or doxy + B12 (n = 10; 4 M 6 F; Fig. 1) was stained for H3K36me3. Representative images are shown with dysplastic foci demarcated by red dashed lines. The mean nuclear optical density of the H3K36me3 stain is expressed as the ratio between the dysplastic region and the adjacent normal tissue for each mouse. Scale bar, 100 µm. Mice that received folate in addition to B12 are represented by open points. Graphs represent the average ± s.d.; ****P < 0.0001 by two-tailed Student’s t-test.
